The present invention, in some embodiments thereof, relates to saccharide-containing active reagents (e.g., glycosylation agents), to conjugates thereof with biomolecules and to methods utilizing these conjugates in e.g., therapeutic applications. More particularly, but not exclusively, the present invention, in some embodiments thereof, relates to saccharide-containing active reagents (e.g., glycosylation agents), to protein conjugates made therefrom and to methods utilizing these protein conjugates in therapeutic applications.
The trafficking of many proteins, and especially lysosomal enzymes, to their target organs, cells and organelles is controlled and enabled by different carbohydrate-specific receptors, such as the mannose or mannose-6-phosphate (M6P) receptors.
In order for proteins to be recognized and transported by carbohydrate-specific receptors, the proteins should be glycosylated with oligosaccharide residues terminating with the appropriate carbohydrate moiety. This is especially important in therapeutic proteins which should be transported to their action site in order to exert their therapeutic benefits.
Of particular importance are the mannose-6-phosphate receptors. The biogenesis of lysosomes, which are key components of the degradative machinery of eukaryotic cells, requires the action of the mannose-6-phosphate receptors (MPRs). Two MPRs, the 300-kDa cation-independent MPR (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR), participate in the intracellular delivery of 50 different lysosomal enzymes to the lysosome by diverting these soluble acid hydrolases from the secretory pathway and delivering them from the trans Golgi network to endosomal compartments. The CI-MPR has also been shown to function in the binding and internalization of ligands at the cell surface. The higher capacity of the CI-MPR as compared with the CD-MPR in sorting lysosomal enzymes to the lysosome is due in part to the ability of the CI-MPR at the cell surface to re-capture lysosomal enzymes that may have been secreted, resulting in their internalization and delivery to endosomal compartments.
In addition to lysosomal enzymes, the repertoire of extracellular M6P-containing ligands has expanded in recent years to include a diverse spectrum of proteins including the precursor form of transforming growth factor and renin, proliferin, granzymes A and B, CD26, and herpes simplex viral glycoprotein D. Several studies have implicated the interaction of these ligands with the CI-MPR at the cell surface as being essential to their activity and/or function, thus expanding the role of the CI-MPR from solely an intracellular protein carrier to a cell surface signaling molecule via its lectin activity.
Oligosaccharides containing M6P have been isolated from lysosomal enzyme mixtures from human skin fibroblasts, mouse lymphoma cells, murine macrophages, and from purified lysosomal enzymes including β-glucuronidase and β-galactosidase. The results of structural analyses indicated that α(1,2)-linked M6P residues are present as terminal or penultimate residues on either, or both, antennary arms of N-linked oligomannosides. In addition, some M6P residues contain N-acetylglucosamine in phosphodiester linkage. The binding properties of the oligomannosides obtained from acid hydrolases have been examined by affinity chromatography on immobilized CI-MPR and CD-MPR. The immobilized receptors bind oligomannosides containing two M6P residues with a greater affinity than those containing a single M6P residue.
As mentioned above, the glycosylation profile of a protein determines its bio-availability and trafficking behavior. The glycosylation profile of a specific protein is highly dependent on its biosynthetic pathway and its expressing platform. Thus, recombinant therapeutic proteins, expressed in various expressing platforms, including bacteria, fungi, plants, and mammalian cells, almost always differ dramatically in their glycosylation sites, glycosylation level and oligosaccharide profile from the original human protein. These differences dramatically diminish the bioavailability and uptake of the recombinant protein by the target cells, resulting in diminished therapeutic potency or necessitating higher doses.
For example, recombinant lysosomal enzymes obtained from different expression systems are often not sufficiently phosphorylated and the level of M6P in the oligosaccharides varies considerably from one expression system to another. The high uptake form of α-galactosidase A is bis-phosphorylated while only 20% of the α-galactosidase A expressed in CHO cells are phosphorylated and only 5% are bis-phosphorylated. Moreover, recombinant proteins expressed in plants, insect cells or yeasts do not have any M6P phosphorylation since these expression systems lack the M6P targeting pathway.
In view of the need to enable the targeting of recombinant therapeutic proteins, and especially of plant recombinant proteins, to carbohydrate-specific receptors, such as the M6P receptor, a few methodologies have been devised to conjugate M6P to proteins, as follows.
U.S. Pat. No. 7,001,994 teaches oxidizing protein glycosides to carbonyls (aldehydes) and reacting these carbonyls with phosphorylated mannopyranosyl oligosaccharides. The phosphorylated mannopyranosyl saccharides are derivatized with a carbonyl reactive substituent, such as hydrazine, which upon reaction with the aldehydes yields a covalent hydrazone bond. This methodology was also exemplified by Cheng et al. (Abstracts of Papers, 232nd ACS National Meeting, San Francisco, Calif., United States, Sep. 10-14, 2006) by attaching a synthetic oligosaccharide ligand bearing M6P residues in the optimal configuration for binding the CI-MPR. In this methodology, the protein undergoes oxidative conditions which are not selective and may cause oxidative damage to additional sites on the protein, including active sites. Such oxidative damage may also promote oxidative stress upon administration.
Beljaars et al. (Liver 2001, 21, 320-328) have synthesized M6P-modified albumin (M6P28-HSA) in order to improve targeting of drugs to hepatic stellate cells. In this publication, it has been shown that the binding of M6P-HSA to the M6P/IGFII receptor is specific. Furthermore, M6P28—HSA was extensively internalized by these cells. Using monensin, a specific inhibitor of the lysosomal pathway, proof was obtained that M6P-HSA is endocytosed via this route. Beljaars et al. have concluded that M6P28—HSA is applicable as a stellate cell-selective carrier for antifibrotic drugs that act intracellularly. The M6P was connected to the albumin protein by phosphorylating p-nitrophenyl-α-D-mannopyranoside, and further reducing the nitro-group to the primary amine. The latter could be coupled to proteins by two methodologies: In the first methodology the p-aminophenyl-sugar was reacted with sodium nitrite under acidic conditions to form the diazonium salt of the derivatized sugar. This diazonium salt readily binds covalently to tyrosine or histidyl residues on the protein. In another optional methodology the p-aminophenyl-sugar is treated with thiophosgen to convert the primary amine to an isothiocyanate group, the latter readily reacts with primary amino groups on the protein, primarily lysine residues.
In a different methodology (see, U.S. patent application Ser. No. 10/024,197) a phosphorylated glucocerebrosidase has been prepared while utilizing isolated GlcNAc phosphotransferase and a phosphodiester α-GlcNAcase. According to this methodology, which mimics natural biosynthetic mechanisms, both enzymes used in the phosphorylation protocol should be produced and isolated, rendering this methodology cumbersome, cost-ineffective and laborious, necessitating the purification of the therapeutic protein from the additional enzymes used in its post-translational modification.
Lee et al. (Glycoconjugate Journal 2006, 23 (5/6), 317-327) have conjugated galactose-6-phosphate (Gal6P) to bovine serum albumin by using glycosides of Gal6P that can potentially generate a terminal aldehyde group, namely Gal6P with a glycerol attached to its anomeric carbon. ω-Aldehydro glycosides were then conjugated to BSA via reductive amination.
Hydrocarbon chains have been used as linkers to oligosaccharides in different biological studies. In WO 92/22662, oligosaccharides having attached thereto an 8-methoxycarbonyloctyl [—(CH2)8CO2CH3] group, and use thereof in a variety of biological applications are taught. This publication also teaches that a PEG chain can be used as the linker.
Distler et al. (J. Biol. Chem. 1991, 266, 21687) have also used an 8-methoxycarbonyloctyl alkyl linker to show the binding specificity of different phosphorylated mannose glycosides to MPR. A series of chemically synthesized oligomannosides that contain M6P residues were utilized as inhibitors of the binding of β-galactosidase to CI-MPR and CD-MPR in order to probe the specificity of each receptor.
Tomoda et al. (Carbohydrate Research 1991, 213, 37-46) have also studied the binding of bovine serum albumin (BSA) derivatized with penta-D-mannose-6-phosphate and have established that the best binding was obtained when the linkage mode between the terminal M6P sugar group and the penultimate sugar residue was α(1→2). Furthermore, they have shown that the length of the sugar chain also affects the binding to the M6P receptor, such that, for example, trisaccharides containing a terminal M6P group were more potent inhibitors than disaccharides.
JP 04210221 describes long chain alkyl D-glucoside-6-phosphates as surfactants for dishwashing and shampoos.
Cowden et al. (U.S. Pat. No. 6,294,521) describe the preparation of sugar phosphates as anti-inflammatory agents. D-mannoside-6-phosphate derivatives wherein the anomeric carbon is derivatized with a long chain hydrocarbon (C8-C16) are used in treating inflammatory diseases, particularly cell-mediated inflammatory diseases.
Similar glucose-6-phosphate derivatives have been described by Jones et al. (Journal of the Chemical Society, Chemical Communications 1994, 11, 1311-12). Dodecyl β-D-glucopyranoside-6-phosphate was used as a novel surfactant possessing a long-chain hydrocarbon tail and a hydrophilic head, consisting of a phosphoryl group covalently linked to a (homochiral) glucose moiety. This compound has been successfully used in miscellar electrokinetic capillary chromatography.
Short PEG chains (n=3) attached at one end to the anomeric carbon of a monosaccharide in a β-conformation and to a fatty acid at the other end are used as detergents in various pharmaceutical and cosmetic applications (see, for example, JP 2766141, JP 2854203, JP 06080686, WO 2006/098415). These publications suggest that such detergents (for example, saccharide-(OCH2CH2)3—NH—CO—CH(C16H33)2) can be used in the preparation of drug delivery systems, mainly liposomes, in combination with other lipids, such as cholesterol and glycerophospholipids.
Similar detergents, which were further used as anchored cryo-protectors, have been prepared by Wilhelm et al. (Liebigs Annalen 1995, 9, 1673-9), using long-chain alcohols (C16) with 0-4 ethoxy spacers. Engel et al. (Journal of Pharmaceutical Sciences 2003, 92(11), 2229-2235) have used the same detergents in the creation of liposomes and have evaluated the interaction of mannose and glucose derivatives of these detergents with Concanavalin A lectin. Engel et al. (Pharmaceutical Research 2003, 20 (1), 51-57) have also improved the uptake of such mannosyl-based liposomes by macrophages, by enhancing the affinity towards mannose receptor. The researchers have suggested that such mannosides with sufficiently long spacer arms are of potential use in receptor-mediated targeting of liposomes made with such detergents.
Millqvist-Fureby et al. have described (Biotechnology and Bioengineering 1998, 59(6), 747-753) the synthesis of ethoxylated glycosides (tetraethylene glycol β-D-glucoside, tetraethylene glycol β-D-xyloside, and methoxy triethyleneglycol β-D-glucoside). These were in turn used as raw materials for the preparation of the above discussed detergents, bearing an additional fatty acid, such as ω-O-oleoyl tetraethylene glycol β-D-glucoside.
These glycoside-PEG-fatty acid detergents were widely investigated for their physical properties (Czichocki et al. Journal of Chromatography, A 2002, 943(2), 241-250; Zimmermann et al. Spectroscopy of Biological Molecules: New Directions, European Conference on the Spectroscopy of Biological Molecules, 8th, Enschede, Netherlands, Aug. 29-Sep. 2, 1999 (1999), 353-354) as well as in various applications, such as biofilm inhibitory and removal agents (JP 2006347941) and for anticaries dentifrices (WO 2006/035821).
PEGylated glycosides, in which the glycosides are conjugated to a small number of ethylene glycol groups, are commercially available although not wide-spread. These glycoside-PEG derivatives are composed of short-chain PEGs (n≦4) and are mainly used to create the surfactants described above by their further conjugation to a hydrophobic moiety, such as a fatty acid.
WO 2005/093422 describes the use of bio-functionalized quantum dots comprising a saccharide derivatized at the anomeric carbon in a β-conformation with a PEG chain (n=6) derivatized at the other end with an alkyl thiol group. These bio-functionalized quantum dots can be used in biological and medical research, imaging and/or therapy applications.
JP 3001381 discloses the use of monosaccharides linked to a PEG chain or an alkyl chain, and further linked to a polysaccharide, such as chitosan, a pullulan, a dextran, mannoglucan, heparin or hyaluronic acid. These moieties were used for delivering drugs following their physical incorporation in the matrices of these polysaccharides, similarly to liposomes.
Andersson et al. (Glycoconjugate Journal 1993, 10(3), 197-201) have prepared glycosides with a linker of a short PEG chain (n=2 or 4) attached to the anomeric carbon (β-conformation). These glycosides were conjugated to proteins through a terminal activated ester. The target application of such glycoside-protein conjugates is as antigens in diagnostic tests or as immunogens. While conjugation with monosaccharides is described, these studies were mainly practiced with oligosaccharides. The PEG linker in all the practiced conjugates was connected to the glycoside via an equatorial bond (β conformation). As noted above, the PEG chains used in this work were relatively short (n=2 or 4).
Biskup et al. (ChemBioChem 2005, 6(6), 1007-1015) have used glycoside with short PEG spacers (n=4) for immobilization of the glycoside onto solid surfaces, followed by interacting the immobilized glycosides with lectins. It appears that the PEG spacers in the studied conjugates were attached to the glycoside anomeric carbon in the β conformation.
Zalipsky et al. (Chemical Communications (Cambridge) 1999, 7, 653-654) have presented a practical approach for preparing galactose-PEG-distearoylphosphatidic acid (DSPA) that retains full lectin binding. Their methodology involved glycosylation of monobenzyl ether-PEG, suitable protection of the sugar hydroxy groups, and debenzylation, followed by enzymatic transphosphatidylation with phosphatidylcholine and final deprotection.
WO 2006/093524 describes compositions comprising antigen-carbohydrate conjugates and methods of immune modulation utilizing these reagents. Thus, ovalbumin, a model antigen, was reacted with the N-hydroxy-succinimide (NHS) group of a bi-functional short hydrocarbon linker to introduce maleimide functional groups. The latter were then reacted with thiol-terminated short PEG chains (n=2-3) attached to saccharides. These saccharides comprise monosaccharides, but mainly high-mannose oligosaccharide. These conjugates were shown to enhance antigen uptake and presentation to T cells, when compared to unmodified ovalbumin. This, in turn, led to improved antigen-specific T cell activation. The bond conformation between the short thiol-terminated PEG chains and the anomeric carbon was both α and β.